The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 39 untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P , 0.001) and UGT2B10 (P 5 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P , 0.001) and UGT2B10 (P 5 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P , 0.001 and P 5 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P 5 0.021) and Hep3B (P 5 0.0068) cells, and increased epirubicin glucuronidation by 85% (P 5 0.057) and 50% (P 5 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P , 0.001 and P 5 0.0048, and P 5 0.017 and P 5 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.
UDP-glucuronosyltransferase (UGT) 2A1 is an important enzyme in the detoxification of polycyclic aromatic hydrocarbons found in cigarette smoke. This enzyme is expressed in aerodigestive tract tissues including lung as both its wild-type and exon 4-deleted splice variant isoforms, with the latter acting as a negative regulator of wild-type UGT2A1 activity. UGT2A1 regulation may also be mediated by microRNA (miRNA). To identify miRNA important in the regulation of UGT2A1, expression analysis in tandem with in silico analysis suggested miR-196a-5p and miR-196b-5p as potential top candidates. Significant reductions in firefly luciferase activity were observed in human embryonic kidney cell line 293 cells cotransfected with the wild-type UGT2A1 3′-untranslated region (UTR)-containing luciferase plasmid and either miR-196a-5p (62%, P = 0.00080) or miR-196b-5p (60%, P = 0.00030) mimics. In pull-down assays, there was a 3.4- and 5.2-fold increase in miR-196a-5p ( P = 0.054) and miR-196b-5p ( P = 0.035), respectively, using the UGT2A1 3′-UTR biotinylated mRNA probe as compared with the β -actin coding region control mRNA probe. UGT2A1 mRNA was reduced by 25% ( P = 0.058) and 35% ( P = 0.023) in H146 and H1944 cells, respectively, after overexpression of the miR196a-5p mimic. A similar 32% ( P = 0.030) and 41% ( P = 0.016) reduction was observed after over-expression of the miR-196b-5p mimic. In H146 cells transfected with miRNA mimic together with a small interfering RNA (siRNA) specific for the UGT2A1 splice variant, a significant reduction in 3-hydroxy-benzo[a]pyrene-glucuronide formation was observed. The miR-196a-5p- and miR-196b-55p-treated cells exhibited reductions of 35% ( P = 0.047) and 44% ( P = 0.0063), respectively. These data suggest that miR-196a-5p and miR-196b-5p play an important role in UGT2A1 regulation within the lung and potentially other aerodigestive tract tissues.
The UDP-glucuronosyltransferase (UGT) family of enzymes is important in the metabolic elimination of a variety of endogenous compounds such as bile acids, steroids, and fat-soluble vitamins, as well as exogenous compounds including many pharmaceuticals. The UGT2B subfamily is a major family of UGT enzymes expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contributes to interindividual variability in UGT2B expression and is a crucial component in predicting patient drug response. In the present study, a high-resolution liquid chromatography–tandem mass spectrometry method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples (n = 62). Concurrent in silico analysis identified eight candidate miRNAs as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant inverse correlation between UGT2B10 and UGT2B15 and one of these candidate miRNAs, miR-485-5p. A near-significant correlation was also observed between UGT2B7 and miR-485-5p expression. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the UGT2B10 3′-untranslated region (UTR), and significant reduction in luciferase activity was also observed for a luciferase vector containing the UGT2B7 3′-UTR; however, none was observed for the UBT2B15 3′-UTR. UGT2B10 and UGT2B7 activities were probed using nicotine and 3′-azido-3′-deoxythymidine, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells upon overexpression of miR-485-5p mimic. This is the first study demonstrating a regulatory role of miR-485-5p for multiple UGT2B enzymes.SIGNIFICANCE STATEMENTThe purpose of this study was to identify novel epigenetic miRNA regulators of the UGT2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that miRNA 485-5p is a novel regulator of UGT2B7 and UGT2B10, which play an important role in the metabolism of many commonly prescribed medications, carcinogens, and endogenous compounds. This study identified potential miRNA-UGT2B mRNA interactions using a novel proteomic approach, with in vitro experiments undertaken to validate these interactions.
Polycyclic aromatic hydrocarbons (PAH) are a class of highly lipophilic and mutagenic carcinogenic compounds found in tobacco smoke and incomplete combustion reactions. Once inside the cell, PAHs are oxidized by cytochrome P450s 1C1 and 1B1 to form reactive species that can form DNA adducts. PAHs are primarily detoxified by the UDP‐glucuronosyltransferase (UGT) family of enzymes, which comprise a major family of phase II metabolism responsible for the detoxification of a variety of endogenous and exogenous compounds including steroids, many drugs and chemotherapeutic agents, and carcinogens. UGT2A1 is an aero‐digestive tract‐expressing enzyme shown to be highly selective toward a broad range PAHs and is expressed in tobacco target tissues, including lung. microRNA (miRNA) are short, noncoding RNA sequences that play a role in translational regulation of gene expression by binding primarily to the 3′ untranslated regions (UTR) of mRNA. Significant (P<0.05 and P<0.001) decreases in luciferase activity were observed in cells over‐expressing heterologous vectors containing the 3′‐untranslated region (UTRs) of UGT2A1 with the miR‐196a and miR‐196b mimics, respectively. Pull‐down assays revealed miR‐196a and miR‐196b bound to the 3′ UTR of UGT2A1 at 3.4‐and 5.3‐fold higher levels, respectively, compared to the coding region control sequence. In the lung cancer cell lines, H1944 and H146, a significant trend is observed when cells were transfected with increasing levels of miR‐196a and miR‐196b (Ptrend=0.0065 and Ptrend=0.0114 for H1944, and Ptrend=0.0219 and Ptrend=0.0091 for H146, respectively). These data indicate that miR‐196a and miR‐196b may be important in regulating UGT2A1 expression in human tissues and may be potential biomarkers for cancer risk in smokers.Support or Funding InformationNational Institute of Environmental Health Sciences (NIEHS) of the National Institute of Health (NIH) to Dr. Philip Lazarus (Grant R01‐ES025460) and the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.